肝细胞癌新型靶向基因治疗体系的构建及应用研究

Construction and Application of a Noval Gene-traget Therapy System in Hepatocellular Carcinoma

构建肝细胞癌(HCC)新型靶向基因治疗体系,并以存活素(survivin)基因为靶点,探讨该体系体外杀伤肝癌细胞的作用。构建由巨细胞病毒(CMV)增强子和甲胎蛋白(AFP)启动子构成的融合启动子(AV)驱动的pcD-NA3.1(-)AV质粒,及含绿色荧光蛋白(GFP)的pcDNA3.1(-)AVGFP和含siRNA-survivin的pcDNA3.1(-)AV-siRNA-survivin质粒,以磷酸钙纳米为载体,导入HepG2、SMMC-7721和Hela细胞中,观察转染效率及体外对肝癌细胞的杀伤效应,并采用流式细胞技术(FCM)检测细胞凋亡率。结果显示:AV启动子可特异性驱动下游基因在HepG2细胞表达。此外,pcDNA3.1(-)AVsiRNA-survivin在HepG2细胞中可有效沉默survivin的mRNA和蛋白表达,并诱导68.8%的细胞死亡,而在Hela和SMMC-7721细胞中无显著作用。生长曲线结果显示,转染了pcD-NA3.1(-)AVsiRNA-survivin的HepG2细胞的生长显著受抑(P<0.05)。结果表明,该新型靶向基因治疗体系可高选择性作用于肝癌细胞,可为临床肝癌的基因治疗提供新的研究思路。

This paper is aimed to explore the efficiency of a noval gene-target therapy system in hepatocellular carcino- ma （HCC） treatment in vitro, by using survivin gene as the target. A new fusion promoter （AV） driving pcDNA3.1 （-）AV plasmid, which contained the cytomegalovirus （CMV） enhancer and alpha-fetoprotein （AFP） promoter, was constructed by molecular biologic method. The eukaryotic expression plasmid peDNA3.1 （-）AVGFP and pcDNA3.1 （-）AVsiRNA-survivin were constructed by cloning and inserting the green fluorescent protein （GFP） and siRNA- survivin sequence into pcDNA3.1（-）AV plasmid separately, Then these two plasmids were transfected into HepG2, SMMC-7721 and Hela cells by using nanoparticles of calcium phosphate. The transfection efficiencies were detected by GFP. Reversed transcript polymerase chain reaction （RT-PCR） and western-blot were used to evaluate the knock- down efficiency of siRNA-survivin. The growth curves and cell death of HepG2 cells transfected with or without pcDNA3, l（-）AVsiRNA-survivin were detected by MTT assay and flow cytometry assay, respectively. The results showed that after transfected with pcDNA3. I（-）AVGFP vector, only HepG2 cells displayed strong GFP signaling, whereas, no GFP was found in Hela cells, suggesting that AV promoter can specifically drive downstream of gene expression in HCC cells. Furthermore, the mRNA and protein expression levels of survivin in HepG2 cells, but not in Hela and SMMC-7721 cells, were significantly silenced after peDNA3, l（-）AVsiRNA-survivin transfection. Final- ly, compared with the control cells, HepG2 cells, which were transfected with pcDNA3.1 （-） AVsiRNA-survivin plasmid, presented 68. 8% cell death, including 38. 680/oo apoptosis and 30. 12% necrosis, and significant cell growth inhibition （P〈0.05）. These findings indicated that this noval gene-target therapy system could specifically target HCC cells with high efficiency, providing a new gene therapy strategy for HCC.